Regulation of ApoB Secretion in Inbred Mouse Strains

描述（由申请人提供）：血浆载脂蛋白B （apo B）和低密度脂蛋白（LDL）水平升高与动脉粥样硬化性冠心病（CHD）的高风险相关，CHD是工业化国家死亡的主要原因。血浆载脂蛋白B水平升高和过量产生载脂蛋白B也是家族性合并高脂血症（FCHL）的主要特征，FCHL是普通人群中普遍存在的疾病。这种流行疾病的遗传基础是异质的和未知的。调节血浆载脂蛋白B水平的基因是导致FCHL表型的基因的逻辑候选基因。载脂蛋白B是肝脏分泌极低密度脂蛋白（VLDL）所必需的，是血浆VLDL和LDL的必需蛋白质成分。血浆载脂蛋白B水平在一定程度上受载脂蛋白B分泌率的控制。利用人载脂蛋白B转基因小鼠模型（HuBTg），我们发现C57BL/6 （B6）和129/Sv（129）背景的HuBTg小鼠血浆载脂蛋白B水平的差异主要是由于肝脏载脂蛋白B分泌率通过转录后调节。进一步的遗传研究发现，6号和4号染色体上的两个新的数量性状位点（QTL）对血浆载脂蛋白B水平有重要影响。这些基因被称为载脂蛋白B调控基因（Apo B regulatory genes, Abrg）。我们的精细定位分析将Abrgl定位在6号染色体上约2.9 Mb的间隔上，Abrg2定位在4号染色体上约12 cm的间隔上。目前的目标是鉴定和表征6号染色体上的Abrgl基因。具体目标是：1。6号染色体上调节血浆载脂蛋白B水平的abgl的定位克隆。将采用几种平行的方法来确定abgl的候选基因。这些方法包括RT-PCR、实时定量PCR和DNA测序分析。研究还将用于确定表达变异的等位基因变异对转录的影响。2. 同源小鼠Abrg基因的鉴定及候选基因的功能研究。我们将通过体外和体内过表达研究对候选变异基因进行功能测试，包括Copsla基因（一种表达变异）。然后将通过敲入和/或敲除基因靶向技术验证abgl基因的最佳候选基因，无论是Cops7a还是其他待鉴定的基因。研究将被设计来描述Abrgl基因的功能作用。总的来说，该基因将成为影响载脂蛋白B的组装和分泌途径的新调控因子。作为载脂蛋白B分泌率的调节因子，该新基因将成为FCHL患者亚群中导致载脂蛋白B过量产生的基因之一的强有力候选基因。它也将成为普通人群中高脂血症患者的药物发现和药理干预的潜在目标。

DESCRIPTION (provided by applicant): Elevated plasma levels of apolipoprotein B (apo B) and low density lipoprotein (LDL) are associated with a higher risk for atherosclerotic coronary heart disease (CHD), a leading cause of mortality in the industrialized world. Elevated plasma apo B levels and overproduction of apo B are also major characteristics of familial combined hyperlipidemia (FCHL), a prevalent disorder in the general population. The genetic basis of this prevalent disease is heterogeneous and unknown. Genes that regulate plasma apo B levels are logical candidates for genes which contribute to the FCHL phenotype. Apo B is required for the secretion of very low density lipoproteins (VLDL) from the liver and is the mandatory protein constituent of both plasma VLDL and LDL. Plasma apo B levels are controlled, in part, by secretion rates of apo B-containing lipoprotein particles. Using the human apo B transgenic mouse model (HuBTg), we have shown that HuBTg mice of C57BL/6 (B6) and 129/Sv (129) background differ in their plasma apo B levels mainly due to hepatic apo B secretion rates via post-transcriptional regulation. Further genetic studies of two mouse strains identified two novel quantitative trait loci (QTL) on chromosomes 6 and 4 which have major effects on plasma apo B levels. These genes are designated Apo B regulatory genes (Abrg). Our fine-mapping analyses have localized the Abrgl to an interval of approximately 2.9 Mb on chromosome 6 and the Abrg2 to a 12-cM interval on chromosome 4. The goals of the current proposal are to identify and characterize the Abrgl gene on chromosome 6. The specific aims are: 1. Positional cloning of the Abrgl on chromosome 6 that regulates plasma apo B levels. Several parallel approaches will be taken to identify candidate genes for the Abrgl. These approaches include RT-PCR, quantitative real-time PCR assays, and DNA sequencing analysis. Studies will also be designed to determine the effects of allelic variations of expression variants on transcription. 2. Characterization of the Abrg genes in congenic mice and functional studies of the Abrgl candidate genes. We will perform functional tests on candidate variant genes, including the Copsla gene (an expression variant), by over expression studies in vitro and in vivo. The best candidate for the Abrgl gene, either Cops7a or another gene to be identified, will then be verified via knock-in and/or knock-out gene-targeting techniques. Studies will be designed to characterize the functional roles of the Abrgl gene. Overall, the gene identified will be a novel regulator affecting the pathways involved in the assembly and secretion of apo B containing lipoproteins. As a regulator of apo B secretion rates, this novel gene will be a strong candidate for one of the genes causative for overproduction of apo B in subsets of FCHL patients. It will also be a potential target for drug discovery and pharmacological intervention in hyperlipidemic patients in the general population.